1. Defining the roles of EGFR pathway components in RCC. Clear cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of genes that contribute to tumor growth and metastasis, including transforming growth factor-alpha (TGF-alpha), a ligand of the epidermal growth factor receptor (EGFR) tyrosine kinase. Autocrine TGF-alpha/EGFR pathway activation in VHL-negative clear cell RCC results in overexpression of the EGFR receptor in RCC compared to normal renal tissue and constitutive activation of certain EGFR pathway effectors, contributing to cell survival, proliferation, and invasiveness. Among the known EGFR effectors, Akt-1 is a serine/threonine kinase activated downstream of phosphatidylinositol 3-kinase (PI3K) that mediates cell survival signals and contributes to oncogenic signaling in RCC cells in culture. Elevated Akt-1 activation is common in RCC, especially in high grade tumors and metastatic disease. MEK-1 is a member of the extracellular signal-regulated kinase (ERK) serine/threonine kinase family that mediates diverse cellular processes including proliferation, survival, differentiation and motility in normal and tumor cells. Constitutive MEK-1 activation has also been demonstrated in RCC tumor tissue relative to surrounding normal tissue, and the level of pathway activation correlates positively with RCC tumor grade and stage. These findings provide a strong impetus to assess the oncogenic contribution of each of these EGFR pathway components functionally in whole animals. To this end, shRNA constructs were used to create stable, VHL-negative RCC cell cultures with diminished expression of EGFR, Akt-1 and MEK-1. We found that each protein contributed significantly to RCC cell proliferation, and that each was required for growth factor-induced extracellular matrix invasion and aggressive tumor growth in mice. Overall our results indicate that each of these signaling components is a critical oncogenic effector in RCC, at or near the level of importance of VHL and HIF-2. Interestingly, matrix invasion was a better predictor of tumorigenicity then cell proliferation, highlighting the value of invasion assays as a predictor of tumorigenesis for RCC and revealing that even partial silencing of Akt-1 or MEK-1 can produce striking changes in RCC cell invasiveness amidst persistent survival and growth signaling. This is particularly relevant to targeted anti-cancer therapies because it suggests that complete silencing of certain genes that may cause unacceptable systemic toxicity may not be required for effective disease control. 2. Preclinical assessment of third party third party inhibitors of the VHL/HIF axis as research tools and to assess their preclinical efficacy. In addition to mRNA knockdown, this project incorporates the study of available pharmacological agents targeting EGFR and other VHL-regulated oncogenic pathways. These include AG 1478 (Tyrphostin), AG 1517 (SU 1571) and gefitinib (Iressa, Astra Zeneca, available through the Cancer Therapeutics Evaluation Program, CTEP). A collaboration with Astra Zeneca has been established to obtain the experimental drug candidates ZD6474, targeting both the EGFR and vascular endothelial growth factor receptor (VEGFR) pathway, and AZD 2171, targeting VEGFR. A similar collaboration with Bayer/Onyx has been established to obtain BAY 43-9006 (sorafenib, Nexavar) and with Pfizer to obtain SU-11248 (Sutent), both of which have shown some success against RCC in early phase clinical trials. In addition to the assays already mentioned, RCC cell lines with and without mediator-targeted shRNA knockdowns or treatment with pharmacological pathway inhibitors will be compared for global changes in gene expression in collaboration with the Microarray Facility at the Advanced Technology Center, NCI. 3. Identification of novel HIF-2 inhibitors. Prior studies have shown that normoxic stabilization of HIF1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis, indicating that it is not the most critical oncogenic substrate of VHL. To isolate compounds that selectively modulate HIF2 for use as research tools and drug development leads, a cell-based high throughput screening assay of the NCI Natural Products Repository was developed in collaboration with CCRs Molecular Target Development Program (MTDP, Drs. James McMahon, Tawnya McKee and Girma Woldemichael). A series of luciferase reporter based reporter plasmids were designed and tested in the 786-0 RCC cell line leading to the selection of a single plasmid for optimization and screening. High throughput screening of the Natural Products Library has already yielded initial leads, which will be chromatographically separated into component structures for individual testing. In parallel, the development of secondary assays for cell viability and HIF target gene expression are underway.